Multi-photon microscopy is an imaging technique in which an excitation laser signal is scanned over a region of interest (i.e., image field) and fluorophores in the image field are excited only when they simultaneously absorb multiple photons of the excitation light. In two-photon microscopy, for example, simultaneous absorption of two photons is required to excite a fluorophore. Multi-photon microscopy is often used to generate fluorescent images of living cells and other microscopic objects and has become an important tool in medical imaging.
Multi-photon microscopy enables imaging of living tissue at depths to about one millimeter (mm). Because longer wavelengths tend to scatter in tissue to a lesser degree than shorter wavelengths, the excitation laser typically provides a signal characterized by an infrared wavelength. To excite the dye to emit a fluorescence photon, two photons of infrared light must be absorbed simultaneously. Infrared excitation light is attractive because it minimizes scattering in the tissue being imaged. In order to create a two-dimensional image of the image field, the laser beam is scanned over the image field while fluorescence light from each point in the region is detected at a camera or photomultiplier tube.
Fluorescent emission from the fluorophores increases quadratically with the intensity of the excitation light. As a result, by strongly focusing the excitation signal, fluorescence can be confined within a narrow focal depth. This gives a depth-of-field resolution comparable to that produced by conventional confocal laser scanning microscopes.
Unfortunately, conventional two-photon microscopy technology suffers from relatively low imaging speeds (typically within the range of 10-20 Hz) because it is difficult to gather sufficient numbers of photons from each pixel at high frame rate. For example, a Ti-Sapphire laser is a commonly used excitation source. Unfortunately, commercially available Ti-Sapphire lasers have an average power of only a few Watts and a repetition rate of around only 80 MHz. This enables a photon collection rate of approximately 100-10,000 photons per pixel, per image frame, and at a frame rate of no more than 10-20 Hz—resulting in a signal-to-noise ratio (SNR) of only 10-100. A higher frame rate could potentially be achieved by simply increasing the scanning speed of the excitation signal (e.g., by 100-fold). Unfortunately, operation at a higher frame rate results in reduced photon collection (to only ˜1-10 photons per pixel per frame). As a result, the deleterious effects of faster scanning on image quality and SNR generally outweigh any potential benefit. Furthermore, in practice, scanning speed is often limited by mechanical and/or optical constraints.
Other comparable microscopy technologies capable of high frame rate are also beset by several disadvantages. Conventional single-photon epifluorescence microscopy suffers from high tissue scattering and low optical sectioning ability. Such disadvantages can give rise to an overlap of photons emitted from different positions into the same pixel of the camera leading to significant image blur, thereby degrading image resolution. Line scanning two-photon microscopy and random access two-photon microscopy with acousto-optic deflector can achieved single focus two-photon scanning at higher frame rate, however at the cost of sacrificing number of pixels being imaged down to a single line of pixels and limited number of arbitrarily selected pixels in focal plane, respectively.
A two-photon imaging system that can provide an image of a region of interest in real time and with improved clarity would be a significant advance in the state-of-the-art.